CHAPTER TWO

2.0 Literature review

2.1 Scientific classification
1 Staphylococci is classified based on the morphological characteristics of the
organism. The taxonomy is based on 16s rRNA sequences (Takahashi et 2009),
land most of the staphylococcal species fall into 11 clusters:
S. aureus group - S. aureus, S. simiae
S. auricularis group — s. auricularis
S. carnosus group -S. carnosus, S. condimenti, S. massiliensis, S. piscifermentans,
S. simulans
S. epidermidis group-S. capitis, S. caprae, S. epidermidis, S. saccharolyticus
S. haemolyticus group - S. devriesei, S. haemolyticus, S. hominis
S. hyicus-intermedius group-S, chromogenes, S. felis, S. delphini, S. hyicus, s.
Iintermedius, S. lutrae, S. microti, S. muscae, S. pseudintermedius, S. rostri, s.
bchleijeri
S. lugdunensis group - s. lugdunensis
S. saprophyticus group-S. arlettae, S. cohnii, S. equorum, s. gallinarum, s. kloosii,
S. leei, S. nepalensis, S. saprophyticus, S. succinus, S. xylosus S. sciurigroup - S.
fleurettiif S. lentus, S. sciuri, S. Stepanovicii,s. vitulinus S. simulans group-S.
simulans
S. warneri group -S. pasteuri, S. warneri
j tna&DCOCCUf, the species of ٦hich are currently the closest known relative‫ ؛‬of
the
Staphylococci (Kloos etal./ 2008).
2.2 Subspecies S'aureus subsp. aureus S. aureus subsp. anaerobius
S٠ capitis subsp. Capitis
s. capitis subsp. urealyticus

S. carnosus subsp. carnosus

S. carnosus subsp. utilis

S. cohnii subsp. cohnii

S. cohnii subsp. urealyticus

S. equorum subsp. equorum

S. equorum subsp. linens

S. hominis subsp. hominis

S. hominis subsp. novobiosepticus

s. saprophyticus subsp. bovis

s. saprophyticus subsp. saprophyticus?

s. schleiferi subsp. coagulans

s. schleiferi subsp. schleiferi

s.sciurisubsp. carnaticus

S. sciuri subsp. rodentium

S. sciuri subsp. sciuri

s.succinussubsp. casei

S. succinus subsp. Succinus

2.3 Biochemical identification

Assignment of a strain to the genus Staphylococcus requires it to be a Gram-

,positive coccus that forms clusters, produces catalase, has an appropriate cell

wall

‫؛‬structure (including peptidoglycan type and teichoic acid presence) and G c ‫؛‬

content of DNA in a range of 30-40 mol% (Ryan and Ray 2004).

Staphylococcus species can be differentiated from other aerobic and facultative

anaerobic. Gram-positive cocci by several simple tests. Staphylococcus spp. are

facultative anaerobes (capable of growth both aerobically and anaerobically).

All

‫ إ‬species grow in the presence of bile salts, it was believed that all species were

I catalase-positive however it is now known that not all Staphylococcus are

coagulase positive (Ryan and Ray 2004).

Growth can also occur in a 6.5% NaCI solution. On Baird Parker medium.

Staphylococcus spp. grows fermentatively, except for s. saprophyticus, which

grows oxidatively. Staphylococcus spp. are resistant to bacitracin (0.04 u disc:

‫ ا‬resistance = clOmm zone of inhibition) and susceptible to furazolidone

(100١g

‫ ؛‬disc: resistance = <15mm zone of inhibition). Further biochemical testing is

needed to identify to the species level (Ryan and Ray 2004).

I When the bacterium divides it divides along two axes, so forming clumps of

‫ ؛‬bacteria. This is as opposed to streptococci which divide along one axis and

so form chains (strep, meaning twisted or pliant).

2.4 Antibiotic resistance

Antibiotic resistance is a serious and growing phenomenon in contemporary

medicine and has emerged as one of the eminent public health concerns of the

21st century: particularly as it pertains to pathogenic organisms (the term is

not especially relevant to organisms which don't cause disease in humans). In

the

, simplest cases, drug-resistant organisms ma٧ have acquired resistance to

first-line ( antibiotics, thereby necessitating the use of second-line agents.

Typically, the

first-line agent is selected on the basis of several advantages including safety,

availability and cost comparatively, the second-line agent is usually broader in

spectrum, possesses a less favourable risk-benefit profile and may be more

expensive or, in more dire circumstances, locally unavailable. In the case of

some

multi-drug resistant (MDR) pathogens, resistance to second and even third-line

antibiotics is sequentially acquired: a case quintessentially illustrated by

Staphylococcus aureus in some nosocomial settings (Donadio et ai, 2010).

) it may take the form of a spontaneous or induced genetic mutation or the

1 acquisition of resistance genes from other bacterial species by horizontal

gene I transfer via conjugation, transduction, or transformation. Many

antibiotic resistance genes reside on transmissible plasmids, facilitating their

transfer.
Exposure to an antibiotic naturally selects for the survival of the organisms

with

the genes for resistance. In this way, a gene for antibiotic resistance may

readily

spread through an ecosystem of bacteria. Antibiotic-resistance plasmids

frequently contain genes conferring resistance to several different antibiotics

(Donadioeta/., 2010).

Genes for resistance to antibiotics, ،ike the antibiotics themselves, are ancient

I (D'Costa et aL, 2011). However, the increasing prevalence of antibiotic-

resistant

bacterial infections seen in clinical practice stems from antibiotic use both

within

human medicine and veterinary medicine. Any use of antibiotics can increase

selective pressure in a population of bacteria to allow the resistant bacteria to

thrive and the susceptible bacteria to die off. As resistance towards antibiotics
becomes more common, a greater need for alternative treatments arises.

However, despite a push for new antibiotic therapies there has been a

continued

decline in the number of newly approved drugs (Donadio et aL, 2010).

Antibiotic

resistance therefore poses a significant problem.

2.5 Mechanisms of antibiotic resistance by staphylococci

(staphylococcal resistance to penicillin is mediated by penicillinase (a form of

P" )lactamase) production: an enzyme that cleaves the p-lactam ring of the

penicillin (molecule, rendering the antibiotic ineffective. Penicillinase-resistant

p-lactam

)antibiotics, such as methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin,

and [flucloxacillin, are able to resist degradation by staphylococcal

penicillinase (^Carter

.(2004 ,.,٥٥
Resistance to methiciJIin is mediated via the mec operon, part of the

Staphylococcal cassette chromosome mec (SCCmec). Resistance is conferred

by

the mecA gene, which codes for an altered penicillin-binding protein (PBP2a or

PBP2') that has a lower affinity for binding p-lactams (penicillins,

cephalosporin,

and carbapenems). ٢his allows for resistance to all p-lactam antibiotics, and

obviates their clinical use during MRSA infections. As such, the glycopeptides

vancomycin is often deployed against MRSA (Menichetti, 2005).

Aminoglycoside antibiotics, such as kanamycin, gentamicin, streptomycin, etc.,

were once effective against staphylococcal infections until strains evolved

mechanisms to inhibit the aminoglycosides' action, which occurs via protonated

amine and/or hydroxyl interactions with the ribosomal RNA of the bacterial

30S

ribosomal subunit (Carter et all, 2004). There are three main mechanisms of
Aminoglycoside resistance mechanisms which are currently and widely

accepted:

Aminoglycoside modifying enzymes, ribosomal mutations, and active efflux of

the

drug out of the bacteria (Carteret all, 2004).

I Aminoglycoside-modifying enzymes inactivate the Aminoglycoside by

covalently attaching either a phosphate, nucleotide, or acetyl moiety to either

the amine or the alcohol key functional group (or both groups) of the antibiotic.

This changes

I the charge or sterically hinders the antibiotic, decreasing its ribosomal

binding affinity. In s. aureus, the best-characterized Aminoglycoside-modifying

enzyme is

‫ إ‬Aminoglycoside adenylyltransferase 4' IA (ANT (4') IA). This enzyme has been

solved by x-ray crystallography. The enzyme is able to attach an adenyl moiety

to
‫ ؛‬the 4' hydroxyl group of many aminoglycosides, including kanamycin and

gentamicin (Sakon et al., 2003).

Glycopeptide resistance is mediated by acquisition of the vanA gene. The vanA

gene originates from the enterococci and codes for an enzyme that produces an

alternative peptidoglycan to which vancomycin will not bind (Sakon et a/.,

2003).

Today, s. aureus has become resistant to many commonly used antibiotics. The

p-

lactamase-resistant penicillins (methicillin, oxacillin, cloxacillin, and

fiucloxacillin)

I were developed to treat penicillin-resistant s. aureus, and are still used as

first-

line treatment. Methicillin was the first antibiotic in this class to be used (it was

I introduced in 1959), but, only two years later, the first case of MRSA was )

reported in England (Jevons, 2001).

‫؛‬Despite this, MRSA generally remained an uncommon finding, even in hospital

settings, until the 1990s, when there was an. explosion in MRSA prevalence in ‫؛‬

hospitals, where it is now endemic (Johnson et all, 2001). 

‫ ا‬MA infections in both the hospitai and community setting are commonly ‫أ‬

treated with non-p-lactam antibiotics, such as clindamycin (a lincosamine) and

co- ! trimoxazole (also commonl٧ known as trimethoprim/sulfamethoxazole)

(Johnson

et at, 2001). Resistance to these antibiotics has also led to the use of new,

broad-

I spectrum anti-Gram-positive antibiotics, such as linezolid, because of its

availability as an oral drug. First-line treatment for serious invasive infections

due to MRSA is currently glycopeptides antibiotics (vancomycin and

teicoplanin).

There are number of problems with these antibiotics, such as the need for

intravenous administration (there is no oral preparation available), toxicity,

and the need to monitor drug levels regularly by blood tests. There are also

concerns glycopeptides antibiotics do not penetrate very well into infected

tissues (this is a particular concern with infections of the brain and meninges

and in endocarditic). Glycopeptides must not be used to treat methicillin-

sensitive s. aureus (MSSA), as outcomes are inferior (Blot et al., 2002).

Because ٥f the high level of resistance to penicillins and because of the

potential ‫ ؛‬for MRSA to develop resistance to vancomycin, the u.s. Centers for

Disease ‫ ا‬Control and Prevention has published guidelines for the appropriate

use of onycin. In situations where the incidence of MRSA infections is known to

be i ‫ أل‬the

th٠،٥entit٧ of the infecting organism is known. After the infection is confirmed

to

be due to a methicillin-susceptible strain of s. aureus, treatment can be changed

I ٤٠ flucioxacillin or even penjcjllin, as appropriate (Biot et al., 2002).

٦ Vancomycin-resistant 5. aureus (VRSA) is a strain of s. aureus that has

become
resistant to the glycopeptides. ٦he first case of vancomycin-intermediate s.

aureus (VISA) was reported in Japan in 1996 (Hiramatsu et al., 2007) ‫ ؛‬but the

first case of s. aureus truly resistant to glycopeptides antibiotics were only

reported in 2002. ٢hree cases of VRSA infection had been reported in the

United States as of 2005 (Menichetti, 2005).

i 2.6Staphylococcus and drug resistance

Beta-lactam antibiotics (e.g. penicillin and cephalosporin) damage bacteria by

inactivating penicillin binding protein (PBPs), enzyme that is essential in the

assembly of the bacterial cell wall (Pinho et al., 2001). Four native penicillin

binding protein are found in Staphylococci, all the four can be inactivated by

these antibiotics (Pinho et al./ 2001). As a result of the weakened cell wall,

treated bacteria become osmotically fragile and are easily lysed. The

Staphylococcal beta- lactamase protein, which cleaves the beta-lactam ring

structure, confers resistant

‫ أ‬to penicillin but not to semj-synthetic penicillin such as methicillin, oxacillin

and
I Resistant t٠ penicillin in staphylococci is mediated by an altered penicillin

binding I protein (PBP2a) which is encode by mecA gene and confers resistant

to most of ! the current beta-lactam antimicrobial agent (Palavecino, 2007). he

plasticity of s.

‫ ا‬aureus has resulted in the emergence of methicillin resistant in different

strains ‫ ا‬with varying degree of antibiotics resistance and virulence pattern

(Holden et at./ 2004 ‫)ا‬. A distinctive feature of methicillin resistant, its

heterogeneous expression,

I borderline resistance, a low level type of resistance to methicillin exhibited by

I strain lacking mecA, was associated with modification in native PBPs, beta- I

lactamase hyper production, or possibly a methicillinase (Diep et al./ 2006).

The resistant phenotype was influence by numerous factors, including mecA and

beta- I lactamase regulatory elements, fern factor and yet to be identify

chromosomal loci (Diep et al7 2006). 

doxaclllin. Acquisition of the mecA gene, which codes for the penicillin binding

protein PBP2a, confers virtually complete resistance to all beta-lactam

antibiotics including the semi-synthetic penicillin (Weese et a/., 2005). PBP2a

has a very low affinity for beta-lactam antibiotics and is thought to aid cell wall

assembly when the normal PBPs are inactivated (Pinhoeta/., 2001).

Staphylococcus aureus is one of the major resistant pathogen of staphylococci

found in the mucous membrane and a human skin of around a third of the

population, extremely adaptable to antibiotic pressure. It is one of the earlier

bacteria to which methicillin resistant was found in 1947, just four years after

the drug started being mass produce (Bullent et a/., 2003). Methicillin was then

the antibiotic of choice, but has since been replacing by oxacillin due to

significant kidney toxicity. Methicillin resistant Staphylococcus aureus (MRSA)

is a strain of Staphylococcus aureus that have evolve resistant to beta-lactam

antibiotics which include penicillins (methicillin, dicloxacillin, nafcillin,

oxacillin e.t.c.) and cephalosporin due to their ability produce the enzyme

penecillinase. Strains unable to produce such enzyme are referred to as

methicillin sensitive s. aureus